In 1975 Kohler and Milstein introduced a procedure for the production of monoclonal antibodies (mAbs) using hybrid cells (hybridomas) which allows the production of almost unlimited quantities of antibodies of precise and reproducible specificity. Conventional antisera, produced by immunizing animals with tumor cells or other antigens, contain a myriad of different antibodies differing in their specificity and properties, whereas hybridomas produce a single antibody with uniform characteristics. The Kohler-Milstein procedure entails the fusion of spleen cells from an immunized animal with an immortal myeloma cell line. From the fused cells (hybridomas), clones are selected that produce antibody of the desired specificity. Each clone continues to produce only that one antibody. As hybridoma cells can be cultured indefinitely (or stored frozen in liquid nitrogen), a constant supply of antibody is assured.
Antibodies are proteins that have the ability to combine with and recognize other molecules, known as antigens. Monoclonal antibodies are no different from other antibodies except that they are very uniform in their properties and recognize only one antigen or a portion of an antigen known as a determinant.
In the case of cells, the determinant recognized is an antigen on or in the cell which reacts with the antibody. It is through these cell antigens that a particular antibody recognizes, i.e. reacts with, a particular kind of cell. Thus the cell antigens are markers by which the cell is identified.
These antigenic markers may be used to observe the normal process of cell differentiation and to locate abnormalities within a given cell system. The process of differentiation is accompanied by changes in the cell surface antigenic phenotype, and antigens that distinguish cells belonging to distinct differentiation lineages or distinguish cells at different phases in the same differentiation lineage may be observed if the correct antibody is available. Initial recognition of differentiation antigens came about through analysis of surface antigens of T-cell leukemias of the mouse and the description of the TL, Thy-1, and Lyt series of antigens. (Old, Lloyd J., Cancer Research, 41, 361-375, February 1981) The analysis of these T-cell differentiation antigens was greatly simplified by the availability of normal T cells and B cells of mouse and man and is relatively advanced. (See Pat. Nos. 4,361,549-550; 4,364,932-37 and 4,363,799 concerning mAb to Human T-cell antigens). Little is known about differentiation antigens displayed on normal and neoplastic cells belonging to other lineages.
This is due to the difficulty of obtaining a ready source of the appropriate normal cell type as well as the vagaries of the art of monoclonal antibodies. The preparation of hybrid cell lines can be successful or not depending on such experimental factors as nature of the innoculant, cell growth conditions, hybridization conditions etc. Thus it is not always possible to predict successful hybridoma preparation of one cell line although success may have been achieved with another cell line.
Progress in defining surface antigens on melanocytes was made possible by the recently discovered technique of culturing melanocytes from normal skin (Eisinger, et al., Proc. Nat'l. Acad. Sci. USA, 79 2018 (March 1982). This method provides a renewable source of proliferating cells for the analysis of melanocyte differentiation antigens.
We recently described our initial analysis of cell surface antigens of human malignant melanoma identified by mouse monoclonal antibodies (Abs) (Dippold et al. Proc. Natl. Acad. Sci. USA 77, 6114-6118 (1980)). This invention relates to a comparable analysis of human renal cancer. Previous work is found in a co-pending patent application Ser. No 297,814 Monoclonal Antibodies To Cell Surface Antigens of Human Renal Cancer and Ser. No. 474,224 Monoclonal Antibodies to Human Renal Cancer Antigens and Method.
Seventeen monoclonal antibodies derived from fusions with spleen cells of mice immunized with established culture lines of renal cancers identified nine cell surface antigenic systems (Ueda, Ryuzo et al, Proc. Natl. Acad. Sci. USA, 78, 5112 August 1981) Six of the systems gp160, S.sub.25, gp120r, gp120nr, gp115, and V.sub.1 represented new antigens not previously described. The other three systems were related to HLA--A, --B, and --C heavy chain and A and B blood group antigens. The most restricted of the newly described antigens were gp160, S.sub.25, gp140 and gp120r. These determinants are found only on cells of renal origin, both normal and malignant, and represent differentiation antigens of human kidney. In addition to the difference in the molecular weight of two of these antigens, gp160, S.sub.25, and gp120r can be distinguished on the basis of differential expression on a panel of cultured renal cancers and normal kidney epithelium and fetal kidney cells. Glycoproteins bearing gp120r share a determinant with renal gp120nr (as indicated by sequential precipitations with monoclonal antibodies that detect gp120r and gp120nr), but gp120nr is found on a broader range of cell types, including fibroblasts and cell lines derived from ovarian, bladder, and colon cancers. The two other systems, gp115 and V.sub.1, have characteristics of broadly occurring differentiation antigens but can be distinguished from each other and from gp120nr by differences in molecular weight, heat stability (V.sub.1 is a heat-stable determinant), and differential expression on cell types of diverse origin.
These systems can be used to characterize and study the nature of renal cancer. Thus, comparison of the S.sub.25 and gp160 phenotypes of different renal cancer cell lines and cultures of normal kidney clearly distinguish these two systems.
The study of renal cancer Old, et al., supra, co-pending Ser. No. 297,814 of melanoma (Dippold, et al Proc. Natl. Acad. Sci. USA 77, 6114 (1980)), has generated a series of mouse Abs that defined 12 new systems of human cell surface antigens. Six of these had been identified as glycoproteins (gp95, gp150, gp160, gp120r, gp120nr, and gp115), three are heat-labile antigens that could not be immunoprecipitated from labeled cell extracts (S.sub.25, M.sub.19, and R.sub.8), and three are heat-stable antigens, presumably glycolipids (O.sub.5, R.sub.24, and V.sub.1). The use of a standard panel of cultured human cells allows, ready comparisons of the reactivity of these monoclonal antibodies in direct serological tests and absorption analysis, and each of the antigenic systems has a distinct pattern of distribution on the cell panel, in terms of both qualitative and quantitative expression of antigens. On the basis of their distribution on different cell types, these 12 antigenic systems can be further classified into three groups: (i) those with characteristics of restricted differentiation antigens (e.g., the renal-specific gp160, S.sub.25, and gp120r antigens and the R.sub.24 antigen of melanoma and melanocytes), (ii) more broadly represented differentiation antigens (e.g., gp95, gp150, M.sub.19, gp120nr, and V.sub.1, and (iii) antigens expressed by every human cell type tested (e.g., O.sub.5 species antigen).
It has also been found that the cell lines derived from stage I renal cancer (confined to the kidney) are gp160.sup.+, whereas cell lines from metastatic renal cancers are gp160.sup.-. Whether this indicates that cancer cells developing metastatic potential lose gp160 expression, or that gp160.sup.+ and gp160.sup.- renal cancers are derived from separate cell lineages is not determined; however, identifying the cell types in normal kidney that express gp160 and other antigens found on renal cancer should give information about the cellular origins of renal cancer.
These serological probes provided by the invention can identify kidney-specific antigens and are of particular interest in the study of kidney structure and function. In addition, some of the more broadly reacting antibodies are useful in studying other tumors --e.g. V.sub.1 which distinguishes astrocytomas from melanomas.
The importance of parallel biochemical and serological characterizations of antigens identified by Abs is illustrated by the analysis of gp120r and gp120nr. Five Abs in this series immunoprecipitated a 120,000-dalton component from labeled extracts of SK-RC-7 renal cancer cells. Pre-clearing the extract with one of these Abs (AB S.sub.6) removed the 120,000-dalton component identified by Ab S.sub.23, indicating that the two Abs were reacting with the same molecule. However, the antigenic determinant detected by Ab S6 and Ab S.sub.23 can be distinguished in M-MHA tests and absorption analysis. Ab S.sub.23 detected a kidney-specific antigen, whereas Ab S.sub.6 reacted with a much broader range of cell types. These results can be explained by postulating two species of gp120 molecules, both carrying the epitope identified by Ab S.sub.6 but only one with the epitope identified by Ab S.sub.23. In agreement with this interpretation, supernatants after clearing with Ab S.sub.23 still reacted with Ab S.sub.6, even though no antigen precipitating with Ab S.sub.23 remained. The epitope identified by Ab S.sub.23 is found only on cells of renal origin, and, because of this restricted distribution, it is referred to as gp120r. The more widely distributed epitope has been designated "nr" to indicate its nonrestricted nature. gp120r and gp120nr may be the products of two separate genes or of a single gene whose product is modified in renal cells. Similar, although less striking, discrepancies in the cellular distribution of antigens identified by different monoclonal antibodies immunoprecipitating gp95 or gp150 molecules have also been explained on the basis of different epitopes being recognized (Dippold, et al. Proc. Natl. Sci. USA 77, 6114-6118 (1980)).